Synthesis of tumor antigenic determinant

ABSTRACT

Processes are provided for the synthesis of the human T-antigenic determinant 2-acetamido-2-deoxy-3-O-(β-D-galactopyranosyl)-α-D-galactopyranoside containing an α-O-glycosidically linked bridging arm O--(CH 2 ) n  COR. The determinant is coupled to carrier molecules to form artificial T-antigens, and to insoluble supports to form T-immunoadsorbents. The artifical T-antigen is shown to elicit a delayed type hypersensitivity reaction as a diagnostic for the presence of cancer in humans.

This application is a divisional of U.S. Ser. No. 918,935, filed Oct.15, 1986 and now pending, which is a continuation of U.S. Ser. No.277,680, filed June 26, 1981, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to syntheses of the human T-antigenicdeterminant and antigens and immunoabsorbents formed therefrom.

Over fifty years ago, Thomsen¹ observed that human red cells, in vitro,could become transformed such that the cells became agglutinated bynormal ABO compatible sera. After extensive investigation, Friedenreich²concluded that Thomsen's agent was a bacterial enzyme which degraded anatural antigenic determinant to liberate the so-called T receptor--astructure which is bound by an agglutinin of general occurrence in humansera. It is now established that the enzyme responsible for thetransformation is a neuraminidase which exposed the T determinant byremoving N-acetyl-α-D-neuraminidic acid residues (α-sialosides) fromcertain sialoglycoproteins³.

In 1966, the structure of this determinant was shown by Kim andUhlenbruck⁴ to be the disaccharide βDGal(1→3)αDGalNAc which, in theglycoprotein, is glycosidically linked to a threonine or serine residue.The T-determinant is now known to occur in a wide variety ofglycoproteins⁵,6.

Recent findings that this structure occurs in tumor-associated antigenshas caused a resurgence of interest in the investigation of this antigenand the corresponding antibodies. In particular, various investigatorshave shown that the T antigen can be demonstrated on tumor cells ofanimal and human origin⁷⁻¹⁰ ; that immediate and delayed typehypersensitivity reactions to the T antigen can be demonstrated inpatients with certain forms of cancer¹¹ ; and that changes in serumanti-T levels can be of diagnostic significance with regard to somecancers¹¹⁻¹².

All of these observations have obvious important clinical implicationsboth potential and realized. However, one major difficulty in developingthe clinical applications is that prior to the present invention, thekey component, a T antigen, was only available from natural sourceschiefly through the laborious extraction of enzymatically treated humanerthrocyte membranes¹³. This procedure affords only relatively smallamounts of material which, because of its origin, is inherentlydifficult to purify and characterize. In addition, for applications suchas delayed hypersensitivity testing in which T antigenic material isinjected into humans, there is the added disadvantage that materialssuch as this, derived from human blood products, carry the risk oftransmission of hepatitis.

The synthesis of a compound,O-β-D-galactopyranosyl(1→3)-O-(2-acetamido-2-deoxy-α-D-galactopyranosyl)-N-Tosyl-L-serine,which contains the terminal disaccharide of the T-antigenic determinant,has been reported¹⁴. However, this compound was never demonstrated tohave utility as the human T-antigenic determinant. It is not readilyapparent whether the compound could be linked to an antigen-formingcarrier molecule and further whether the resulting conjugate, if formed,would function as an artificial T-antigen. In fact it would be predictedthat the unnatural highly antigenic N-tosyl group present in theaglycone moiety would cause antigens from this compound to beimmunochemically dissimilar from the natural T-antigen.

In U.S. Pat. No. 4,137,401 issued to Lemieux, Bundle and Baker, abridging arm is disclosed O-β-glycosidically linked to aldose moieties.The bridging arm has the structure O--R--COR" where R is an aliphatichydrocarbon moiety having 3-17 carbon atoms and R" is H, OH, NH₂, NHNH₂,N₃ or a lower alkoxy group. The bridging arm enables one to linkcarbohydrate antigenic determinants to carrier molecules or solidsupports to produce artificial antigens and immunoabosrbents. It shouldbe appreciated that the reaction conditions set forth in the referencefor attaching the bridging arm to the aldose moiety are those which willproduce a β-D-anomeric glycosidic linkage. An α-D-anomeric bridging armis desired in the T-antigenic determinant.

In U.S. Pat. No. 4,195,174 issued Mar. 25, 1980, to Lemieux andRatcliffe, processes are provided for the syntheses ofO-acylated-2-azido-2-deoxy glycosyl halides. These halides can beconverted to O-acylated-2-azido-2-deoxy glycosides. In particular thepatent reports the synthesis of3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-galactopyranosyl chloride and itsreaction with alcohols, including an aglycone bridging arm, to form3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-galactopyranosides.

SUMMARY OF THE INVENTION

As a solution to the problem of availability of T-antigenic material, wenow wish to disclose a process which allows for the chemical synthesisof the T-antigenic determinant and thereby artificial T-antigens inlarge amounts. Of particular importance in the present invention is thedemonstration that a synthesized artificial T-antigen can elicit adelayed type hypersensitivity (DTH) reaction diagnostic for the presenceof cancer. This invention represents the first example of a haptenspecific DTH response to an artificial antigen wherein the primaryimmunizing antigen was of natural origin.

In addition to the preparation of these artificial antigens,methodologies are also presented for the preparation ofT-immunoadsorbents useful, for example, in the isolation ofanti-T-antibodies.

In accordance with the process of the present invention,3,4,6-tri-O-acyl-2-azido-2-deoxy-α-D-galactopyranosyl bromide is reactedwith a monohydroxycarboxylate of the general formula HO(CH₂)_(n) CORwhere n=3-19 and R is an alkoxy or aryloxy protecting group, in thepresence of R₄ 'NBr where R' is a lower alkyl group, in a suitablesolvent. This product of this reaction is thereafter, in any order,reduced and N-acetylated to convert the azide group to an acetamidogroup, and de-O-acylated, to produce an O-α-glycoside having thestructure: ##STR1##

In accordance with another aspect of the present invention, the aboveO-α-glycoside is formed by reactingO-acylated-2-azido-2-deoxy-β-D-galactopyranosyl chloride with amonohydroxycarbonate of the general formula HO(CH₂)_(n) COR where n=3-19and R is an alkoxy or aryloxy protecting group, in the presence of thepromoter mercuric cyanide, in a suitable solvent. The product of thisreaction is thereafter, in any order, reduced and N-acetylated toconvert the azide group to an acetamido group, and de-O-acylated, toproduce the above O-α-glycoside.

The O-α-glycoside is thereafter selectively blocked at the4,6-O-positions and condensed with a2,3,4,6-tetra-O-acyl-α-D-galactopyranosyl halide in the presence of apromoter, to form, after deblocking, the T-antigenic determinant haptenhaving the structure: ##STR2##

The bridging arm --O(CH₂)_(n) COR permits attachment, through an amidelinkage of the carbonyl group, to a variety of insoluble or solubleaminated or amine-containing supports to yield artificial antigens andimmunoadsorbents of the T-antigenic determinant. The bridging armembodied in the present invention is 8-methoxycarbonyloctanol, howevervariations in the length of the alkyl chain or in the nature of thealkoxy group would be obvious to a skilled chemist and would not beexpected to alter the utility of the above monosaccharide ordisaccharide products of this invention.

It will be recognized that the abovedescribed processes produce thedesired O-α-anomeric linkage with the bridging arm of the monosaccharideproduct, and the desired O-β-anomeric linkage between the two sugars ofthe disaccharide product. The use of the α-D-galactopyranosyl bromide asopposed to the β-D-galactopyranosyl chloride as the starting material ispreferred since the former is simpler to prepare and yet offers the sameyield of the desired products as does the β-D-galactopyranosyl chloride.

In arriving at the processes of the present invention it was found thatthe α,β ratios of the 2-azido glycosides formed vary unpredictably withnot only the particular 2-azido-galactopyranosyl halide startingmaterial, but also with the particular reaction conditions used and theparticular alcohol being condensed with the halide. For instance, if theconditions of mercuric cyanide as a promoter with α-D-galactopyranosylbromide are used, the undesired β-D-galactosaminide is formed in about75% yield. If mercuric cyanide and mercuric bromide are used, again a70-75% yield of the β-D-galactosaminide is obtained. Use of silvercarbonate with catalytic silver trifluoramethanesulfonate as a promoterwith 8-methoxycarbonyloctanol and either the β-D-galactopyransoylchloride or the α-D-galactopyranosyl bromide, as reported in U.S. Pat.No. 4,195,174, leads to more of the β-glycoside than the α-glycoside.The silver carbonate, silver trifluoromethanesulphonate conditions canlead to the production of α-D-galactosaminides when the alcohol is asugar, however these same conditions yield the undesired β-glycosidelinkage when the alcohol is the aliphatic alcohol bridging arm.

In accordance with another aspect of the invention a method is providedfor detecting a delayed types hypersensitivity reaction to theartificial T-antigen. The method comprises injecting intradermally inthe human body the T-antigenic determinant (B) attached through an amidelinkage of the carbonyl group of the bridging arm to a non-toxic solubleaminated or amine-containing antigen-forming carrier molecule, andthereafter observing a delayed type hypersensitivity reaction by thehuman body. The preferred artificial antigen is8-methoxycarbonyloctyl-2-acetamido-2-deoxy-3-O-(β-D-galactopyranosyl)-α-D-galactopyranosideattached to human serum albumin. The amount of the T-antigenicdeterminant incorporated on the carrier molecule is preferably in therange of about 7 to 16 equivalents/mole.

DESCRIPTION OF THE DRAWING

FIGS. 1-8 on the formula sheets show the structural formulas and namesfor compounds referred to by number in the specification.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The first step in the process for the preparation of the desiredT-antigenic determinant is to attach the monohydroxy carboxylate of thegeneral formula HO(CH₂)_(n) COR, where n=3-19 and R is an alkoxy oraryloxy group to an O-acylated-2-azido-2-deoxy-D-galactopyranosyl halidewith an α-D-anomeric linkage. In Example I herebelow, the particular8-methoxycarbonyloctanol linking arm is α-linked to3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-galactopyranosyl chloride (1) inthe presence of mercuric cyanide promotor in a suitable solvent. InExample II herebelow, 8-methoxycarbonyloctanol is α-linked to3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-galactopyranosyl bromide (2) inthe presence of a halide ion source R₄ 'NBr where R' is a lower alkylgroup, in a suitable solvent. In each case the O-α-glycoside formed bythese reaction conditions is thereafter reduced and N-acetylated toconvert the azide group to an acetamido group, and de-O-acylated toproduce the O-α-glycoside having the structure: ##STR3## Thede-O-acylation step may alternatively precede the reduction andN-acetylation step.

With the bridging arm 8-methoxycarbonyloctanol n=8 and R is a methoxygroup. However, variations in the length of the aliphatic chain betweenabout 3 and 19 will not significantly alter the bridging arm. The Rgroup is selected from alkoxy and aryloxy protecting groups such asmethoxy, ethoxy, propyloxy, butoxy and phenyloxy. In the aboveO-α-glycoside product, the initial R protecting group can be replaced,to facilitate coupling to a carrier or support. Suitable groups includeNHMH₂, N₃ and OH.

The halide ion source R₄ 'NBr, used in Example II is tetraethylammoniumbromide. Alternatives to the ethyl groups are lower alkyls such asethyl, propyl or butyl groups.

In both Examples I and II the particular galactopyranosyl halidestarting material is O-acetylated at the 3,4,6-O-hydroxy positions.Alternative protecting groups include acyl groups such as propionyl andbenzoyl groups.

The solvent used in the abovedescribed processes is one which is capableof dissolving the starting materials at a level to provide sufficientconcentration of these materials to react. The solvent is also selectedto be substantially inert to the reaction taking place. The solvent usedin Example I is dry benzenenitromethane. In Example II the solvent isdichloromethane. Alternative solvents having the abovedescribedproperties will be evident to persons skilled in this art.

EXAMPLE I 8-Methoxycarbonyloctyl2-acetamido-2-deoxy-α-D-galactopyranoside (3)

A solution of 3,4,5-tri-O-acetyl-2-azido-2-deoxy-β-D-galactopyranosylchloride (1, 14.0 g) in benzene (20 mL) was added to a mixture of8-methoxycarbonyloctanol (8.46 g), mercuric cyanide (11.77 g), Drierite(42 g) and dry benzenenitromethane 1:1 (v/v) (225 mL). This mixture wasstirred at 45° to 50° for 72 h, at which time the solids were removed byfiltration through a Celite pad. The filtrate was concentrated to asyrup and dissolved in dichloromethane (200 mL). The resulting solutionwas washed with water (2×100 mL), dried, filtered, and concentrated to asyrup (17.8 g). Without further purification, this material wasdissolved in acetic acid (50 mL) and hydrogenated at 100 psi at roomtemperature in the presence of 5% palladium on charcoal for 4 h. Aceticanhydride (2 mL) was added and the catalyst was removed by filtration.Reduction of the azido group may also be achieved with hydrogen sulfidein a basic solution or with metallic zinc. The filtrate was diluted withtoluene and evaporated to a foam (17 g). Removal of the O-acetyl groupsby transesterification using a catalytic amount of sodium methoxide inmethanol (30 mL) followed by removal of the sodium ions with an acidresin and evaporation gave a foam (10.1 g). Crystallization from hotwater provided pure 8-methoxycarbonyloctyl2-acetamido-2-deoxy-α-D-galactopyranoside (3, 5.1 g) in an overall 35%yield, m.p. 138°-140°, [α]_(D) ²⁵ +130.4° (c, 1.25, methanol); ¹ Hnmr(D₂ O) δ: 4.95 (d, 1H, J₁,2 =3.0 Hz, H-1), 2.12 (s, 3H, NAc); ¹³ Cnmr(CH₃ OD) δ: 98.6 (C-1), 62.7 (C-6), 51.6 (C-2).

Anal. calcd. for C₁₈ H₃₃ N₁ O₈.1/2H₂ O: C, 53.98, H, 8.56; N, 3.50;found: C, 53.98; H, 8.31; N, 3.46.

8-Methoxycarbonyloctyl 2-acetamido-2-deoxy-β-D-galactopyranoside

The mother liquor from the above mentioned crystallization appeared tocontain additional quantities of (3) along with an about 20% overallyield of the β-D-anomer. Consequently, the material was acetylated forchromatography on a silica gel column developed withhexane-ethyl-acetate-ethanol (6:4:1). The fraction which appeared topossess the β-anomer of O-acetylated (3) was de-O-acetylated in theusual manner using sodium methoxide in methanol. The productcrystallized readily from methanoldiethyl ether, m.p. 178°-179.5°,[α]_(D) ²⁵ -3.2° (c, 0.9, methanol); ¹ Hnmr (CD₃ OD) δ: 4.40 (d, 1H,J₁,2 =7.6 Hz, H-1), 2.01 (s, 3H, NAc); ¹³ Cnmr (CD₃ OD) δ: 102.9 (C-1),62.3 (C-6), 54.2 (C-2).

Anal. calcd. for C₁₈ H₃₃ N₁ O₈ : C, 55.22; H, 8.50; N, 3.58 found: C,55.44; H, 8.73; N, 3.62.

This compound is more readily prepared via the use of3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-galactopyranosyl bromide (2).

The α-bromide compound (2) may also be used to synthesize theα-glycoside (3). This is exemplified below.

EXAMPLE II

The α-bromide (2) (17.2 g) was stirred at ambient temperature with amixture of 8-methoxycarbonyloctonal (16.0 g), tetraethylammonium bromide(9.6 g) and 4A° molecular sieves (60.0 g), in dichloromethane (75 mL)for 4 days. At that time the mixture was centrifuged to remove solidsand the supernatant diluted with dichloromethane (400 mL) and washedwith water (2×100 mL). Drying and evaporation of the organic layer gaves syrup (27.2 g). Treatment of this residue with metallic zinc (30.0 g)in acetic acid (75 mL) and acetic anhydride (25 mL) for 0.5 h, effectedreduction of the azide and acetylation of the amine to provide the crudeblocked α-D-galactosaminide. Dilution of the reaction mixture withdichloromethane (100 mL), filtration and evaporation gave a residuewhich was dissolved in dichloromethane (200 mL) and washed successivelywith water (50 mL), saturated sodium bicarbonate (100 mL) and water(2×100 mL). The organic layer was dried and evaporated to give syrupwhich was de-O-acetylated and crystallized as described above in ExampleI to provide 8-methoxycarbonyloctyl2-acetamido-2-deoxy-α-D-galactopyranoside (3) (5.6 g).

In the next step of the process, the monosaccharidic O-α-glycoside isselectively blocked at the 4,6-O-hydroxyl positions to give a compoundhaving the C-3 hydroxyl group as the only free hydroxyl group. Thepreferred blocking group is an acetal such as benzylidene. With thepreferred bridging arm this step yields8-methoxycarbonyloctyl-2-acetamido-4,6-O-benzylidene-2-deoxy-α-D-galactopyranoside(4).

EXAMPLE III 8-Methoxycarbonyloctyl2-acetamido-4,6-O-benzylidene-2-deoxy-α-D-galactopyranoside (4)

Compound (3) (5.0 g) was dissolved in N,N-dimethylformamide (20 mL)containing α,α-dimethoxytoluene (8 mL) and p-toluene-sulfonic acid (0.10g). This mixture was heated to 50° for 5 h, at which time triethylamine(0.5 mL) was added and the solution was taken to dryness under vacuum togive an amorphous glass (5.2 g) which was extracted with pentane (2×50mL). Crystallization of this solid from ethyl acetate-pentane gave thetitle compound (4.5 g), m.p. 145°-146°, [α]_(D) ²⁵ +101° (c, 1,chloroform); ¹ Hnmr (CDCl₃) δ: 5.74 (d, 1H, J₁,2 =3.5 Hz, H-1), 2.02 (S,3H, CH₃). ¹³ Cnmr (CDCl₃) δ: 101.2 (CHPh), 98.6 (C-1), 62.9 (C-6), 50.5(C-2).

Anal. calcd. for C₂₅ H₃₇ N₁ O₈ : C, 62.61; H, 7.78; N, 2.92; found C,62.65; H, 7.75; N, 2.79.

In the final step of the synthesis the selectively blockedmonosaccharide is reacted with a2,3,4,6-tetra-O-acyl-α-D-galactopyranosyl halide in the presence of thepromotor in a suitable solvent, to form a β-D-anomeric glycosidiclinkage between the halide and the 3-hydroxyl group of the blockedglycoside. Subsequent removal of the O-protecting groups on both sugarsyields the T-antigenic determinant hapten having the structure ##STR4##where n=3-19 and R is an alkoxy or aryloxy group.

The preferred acyl protecting groups on the galactopyranosyl halide areacetyl groups. The preferred promotor is mercuric cyanide, since it hasbeen found effective in forming the desired O-β-glycosidic linkage withthese particular reagents. The preferred solvent is drybenzene-nitromethane, however other inert solvents which will dissolvethe starting materials to a sufficient extent, may be chosen.

EXAMPLE IV The Synthesis of the T Antigenic Determinant Hapten8-Methoxycarbonyloctyl2-acetamido-2-deoxy-3-O-(β-D-galactopyranosyl)-α-D-galactopyranoside (6)

A solution of 2,3,5,6-tetra-O-acetyl-α-D-galactopyranosyl bromide (0.315g) in benzene (2 mL) was added to a mixture of compound (4) (0.30 g),mercuric cyanide (0.18 g) anhydrous calcium sulfate (0.97 g) and drybenzene-nitromethane 1:1 (v/v) (50 mL). This mixture was stirred at 50°for 3 h, at which time another portion of the bromide (0.05 g) was addedand the reaction was continued for an additional hour. The solids wereremoved and the filtrate was diluted with dichloromethane (100 mL),washed with water (2×50 mL) and dried. Solvent removal left a foamyproduct (5, 0.50 g) which resisted crystallization. The material wasdissolved in dichloromethane (5 mL) and 90% aqueous trifloroacetic acid(1 mL) was added. (Aqueous acetic acid or hydrogenation can also be usedfor deprotection of the 4 and 6 hydroxyl groups.) After 2 min. at roomtemperature, toluene (5 mL) was added and then the solvent removed undervacuum at 30°. The residue was applied to a column (20×1.5 cm) of silicagel (40 g) which was eluted with benzene-ethyl acetate-ethanol (3:3:1).The main fraction provided a syrup (0.30 g) [¹³ Cnmr (CDCl₃): 97.7(C-1), 101.7 (C-1')] which was de-O-acetylated with catalytic sodiummethoxide in methanol followed by removal of the sodium ions with anacid resin. Filtration and evaporation gave a foam (0.175 g, 51% yield),which was one homogenous spot by tlc developed with isopropanol-ammoniumhydroxide-water (v/v) 7:1:2, and was crystallized from n-butanol-ethanolto give pure 8-methoxycarbonyloctyl2-acetamido-2-deoxy-3-O-(β-D-galactopyranosyl)-α-D-galactopyranoside(6), m.p. 208°-210°, [α]_(D) ²⁵ +92.7° (c, 1.05, water); ¹ Hnmr (D₂ O)δ: 4.91 (d, 1H, J₁,2 3.5 Hz, H-1), 4.49 (d, 1H, J_(1'),2' 6.25 Hz,H-1'), 2.05 (s, CH₃). ¹³ Cnmr (CD₃ OD) δ: 98.1 (C-1), 105.7 (C-1').

Anal. calcd. for C₂₄ H₄₃ N₁ O₁₃.H₂ O: C, 50.42; H, 7.93; N, 2.45. found:C, 50.62; H, 8.00; N, 2.53.

The activity of compound (6) as the T antigenic determinant was shown(a) by demonstrating inhibition of the natural T agglutinins withcompound (6); (Example V) (b) by production of anti-T antibodies with anartificial T antigen (Example VI and VII) (c) by preparation of anefficient T immunoadsorbent from compound (6) (Example VIII) and (d) bythe preparation of T agglutinable erythrocytes by covalently couplingthe T hapten to erythrocytes (Examples IX and X).

EXAMPLE V Inhibition of human anti-T by compound (6)

The agglutination of neuraminidase treated O erythrocytes²⁰ by humananti-T (titre 1/32) was totally inhibited by compound (6) at aconcentration of 1 mg/mL whereas methyl β-D-galactopyranoside and8-methoxycarbonyloctyl 2-acetamido-2-deoxy-α-D-galactopyranoside (3)showed no inhibition at this concentration.

The utility of compound (6) for the preparation of artificial antigenswas shown by attaching the disaccharide, through an amide linkage of thecarbonyl group of the bridging arm, to a soluble carrier. In Example VI,compound (6) is converted to the acyl hydrazide by treatment withhydrazine hydrate and then attached by the previously described acylazide coupling method¹⁹ to soluble carriers to give artificialT-antigens. Suitable antigen forming carrier molecules are recognized,by persons skilled in the art, to be soluble, high molecular weight,aminated or naturally amine-containing compounds. Exemplary carriersinclude proteins, glycoproteins and polysaccharides.

EXAMPLE VI Preparation of the 8-Hydrazinocarbonyloctyl2-acetamido-2-deoxy-3-O-(β-D-galactopyranosyl)-α-D-galactopyranoside (7)

Compound (6) was dissolved in hydrazine hydrate and left to stand for 2h. At that time, the solvent was removed by co-evaporation withbutanol-water 1:1 (v/v) 3×5 mL) to provide the hydrazide (7) used inpreparing artificial antigens and immunoadsorbents as described below.

EXAMPLE VII Preparation of a Synthetic Antigen from8-Hydrazinocarbonyloctyl2-acetamido-2-deoxy-3-O-(β-D-galactopyranosyl-α-D-galactopyranoside (7)

A suspension of compound (7) (0.109 g) in dimethylformamide (1.5 mL) wascooled to near -20° C. under an inert atmosphere and to this was addeddioxande 4.5N in HCl (186 μL) and tertiary-butyl nitrate (50 μL). Theresulting solution was stirred while maintaining cooling for 2 h. Atthat time, the reaction was quenched by the addition of sulfamic acid(0.01 g), dissolved in dimethylformamide (0.408 mL) and stirring andcooling was continued for 15 min. This mixture was then added directlyto a solution of human serum albumin (HSA) and 0.2N aqueousN-ethyldiethanolamine (36 mL, pH 9.03), cooled to 0°-4° C. After 20 h,this mixture was dialyzed against water to remove salts and unreactedreagents and lyophilized to provide the artificial antigen. Acarbohydrate determination showed an incorporation of 13 hapten groupsper HSA molecule.

The value of incorporation can be varied through a range from 6 to 30 bydecreasing or increasing the amount of compound (7) used in relation tothe amount of HSA used. Preferred hapten incorporation values range fromabout 7 to 16 equivalents/mole. Alternate buffers can be used such as aborate buffer, and alternate carrier molecules may be utilized.

    ______________________________________                                        Antigen                   Hapten Incorporation                                Designation                                                                            Carrier          Equiv/mole (n)                                      ______________________________________                                        T-BSA    Bovine Serum Albumin                                                                           22                                                  T-Hb     Horse Hemoglobin 20                                                  T-HSA    Human Serum Albumin                                                                            13                                                  T-IgG    Human Immunoglobulin G                                                                         18                                                  ______________________________________                                    

The utility of compound (6) for the preparation of an immunoabsorbent ofthe T-antigenic determinant is illustrated in Example VIII. Thedeterminant is attached through an amide linkage of the carbonyl groupof the bridging arm to an insoluble aminated or amine-containingimmunoadsorbent-type support. The properties desirable inimmunoadsorbent-type supports are well known in the art (see for exampleAffinity Chromatography, C. R. Lowe and P. D. G. Dean, John Wiley &Sons, London, 1974). Exemplary supports include derivatives ofcellulose, polystyrene, synthetic poly-amino acids, crosslinkeddextrans, polyacrylamide gels, porous glass and agarose.

EXAMPLE VIII Preparation of an Effective Immunoadsorbent from8-Hydrazinocarbonyloctyl2-acetamido-2-deoxy-3-O-(β-D-galactopyranosyl)-α-D-galactopyranoside (7)

A solution of the acyl azide obtained from compound (7) (7.7 mg) indimethylformamide (0.5 mL) as described in Example VI has added to aslurry of calcined diatomaceous earth (100-120) mesh, (20 g), which hasbeen silylaminated²¹, in acetonitrile (30 mL) at 4° C. After standingovernight, the solid was filtered and washed with methanol.N-acetylation of unreacted amines was achieved with acetic anhydride inmethanol. Filtration and drying gave the T immunoadsorbent. Haptenincorporation was 0.3 M/g.

Human O sera (anti-T titre 1/32) adsorbed with 100 mg/mL of Timmunoadsorbent showed after adsorption no anti-T activity.

EXAMPLE IX Preparation of 8-Hydroxycarbonyloctyl2-acetamido-2-deoxy-3-0-(β-D-galactopyranosyl)-α-D-galactopyranoside (8)

The ester compound (6) (0.17 g) was treated with 0.1N NaOH (4 mL) atroom temperature for 2 h. The solution was then deionized with an acidicion exchange resin and the solvent removed to provide the title acid(8).

EXAMPLE X Preparation of T haptenized cells

One mL of packed red blood cells (type 0 Le^(a+b-)) was washed threetimes with fine volumes of buffer (0.01M N-ethyldiethanolamine, 0.15MNaCl, pH 6.0). Three-quarters of a mL of these washed cells was thensuspended in 1.75 mL of the same buffer to give a 30% suspension, whichwas cooled in a 12×75 mm test tube.

Acid derivative (8) (5 Mg) and1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (3 Mg) weredissolved in 150 μL of 0.15M NaCl. This solution was held at roomtemperature for five minutes, and then added to the red blood cellsuspension at 4° C. The reaction mixture was incubated at 4° C. withgentle rocking for two hours, and reagents removed by washing five timeswith five volumes of phosphate buffered saline.

Haptenization was confirmed by serological testing, which showedagglutination of the treated erythrocytes by human anti-T antibodies.

In the past the immune response has been considered to have twoindependent forms, one an antibody mediated response in which pathogensare neutralized by specific antibody molecules synthesized by the Blymphocytes and the other a cell mediated immunity based on theprotective functions of thymus derived (T) lumphocytes. It is now knownthat this view is a gross oversimplification as it is apparent thatthere is a great deal of interaction between these branches of theimmune system and that the adaptive reaction of an individual to anantigen challenge involves a complex network of interacting cells andsoluble cell products. At present the understanding of the factorscontrolling this interaction is quite limited. With regard to delayedtype hypersensitivity reactions, "a cell mediated" response, it is knownthat for a given hapten many factors such as carrier type and haptenincorporation will determine in an as yet unpredictable manner if ahapten specific DTH reaction will take place²².

In the case of the T antigen it has been observed that although allhealthy individuals possess anti-T agglutinins in their sera they havein general no DTH reactions to intradermal injection of T antigen¹¹. Incontrast individuals with certain forms of carcinoma particularly breastcarcinoma will show a DTH reaction to T antigen¹¹. Thus this reaction tothe natural T antigen is strongly indicative of the presence ofcarcinoma and is of great diagnostic significance. Evidence inindividuals with diagnosed cancer for DTH reactions to a product of thepresent invention, an artificial T antigen, T HSA, is presented below.

EXAMPLE XI Demonstration of Delayed Type Hypersensitivity DTH to a T-HSAartificial antigen in Patients with Metastatic Breast Tumors

Patients

The study group (23 patients) was comprised of post-operative stage IVmetastatic breast cancer patients currently under treatment at the W. W.Cross Cancer Institute, Edmonton, Alberta, Canada. Informed consent wasobtained from all the individuals who volunteered to participate in thisstudy.

Antigens

Conjugates of human serum albumin (HSA) with the T hapten were preparedunder aseptic conditions as described above. For use in the human body,the antigen-forming carrier molecule should be non-toxic. Four differentincorporations n=7, 12, 14 and 22 were examined. The HSA used wasprepared by Cohn fractionation of human plasma in accordance with therequirements of the U.S. Food and Drug Administration. All patients alsoreceived HSA, which was processed as described for the preparation ofT-HSA antigen except that no hapten was used in the reaction mixture.

Administration of the Antigen

Both T-HSA and HSA were injected intradermally in separate sites on theupper arm in a total volume of 0.1 mL saline. Antigen concentrationswere 100 and 200 Mg/mL (10 and 20 Mg of antigen per injection).

Delayed Type Hypersensitivity Reactions

A positive reaction was taken as erythema, with or without induration,of greater than 5 mm diameter at 24 h. Where doubt existed weight wasgiven to induration. Positive responses to T-HSA varied from 8 to 20 mm.In some cases positivity to T-HSA was confirmed by skin punch biopsieswhich showed perivascular lymphocyte infiltration.

Results

Of the patients tested none showed positively to HSA and 16 (70%) showeda DTH reaction to T-HSA (n=12 or 14). Of the patients giving a positivereaction the response was greater at the highest antigen concentration.The DTH response was also effected by hapten incorporation with thegreatest response being to antigens with `n` values being between 12 and14. Those patients not responding to T-HSA were found to have recentlyundergone (within 3 months) various therapy programs and therebypossibly rendered anergic to the T antigen. Such patients were excludedfrom the study in its later stages.

While the present invention has been disclosed in connection with thepreferred embodiment thereof, it should be understood that there may beother embodiments which fall within the spirit and scope of theinvention as defined by the following claims.

REFERENCES:

1. O. Thomsen, Z. Immun.-Forsch., 52 (1927) 85-107.

2. V. Friedenreich, The Thomsen Hemagglutination Phenomenon, Levin &Munksgaard, Copenhagen (1930).

3. C. M. Chu, Nature, 161 (1948) 606-607.

4. Z. Kim and G. Uhlenbruck, Z. Immun.-Forsch., 130 (1966) 88-99.

5. R. R. Race and R. Sanger, Blood Groups in Men, 6th ed., BlackwellScientific Publications, Oxford (1978) 486-487.

6. Vaith and G. Uhlenbruck, Z. Immun.-Forsch., 154 (1978) 1-14.

7. P. J. Klein, R. A. Newman, P. Muller, G. Uhlenbruck, H. E. Schaefer,K. J. Lennartz and R. Fischer, Klin, Wschr., 56 (1978) 761-765.

8. D. R. Howard, Vox. Sang., 37 (1979) 107-110.

9. R. A. Newman, P. J. Klein and P. S. Rudland, JNCI 63, (1979)1339-1346.

10. J. H. Anglin, Jr., M. P. Lerner and R. E. Nordquist, Nature, 269(1977) 254-255.

11. G. F. Springer, P. R. Desai, M. S. Murthy, H. Tegtmeyer and E. F.Sanlon, Prog. Allergy, 26 (1979) 42-96 and references contained therein.

12. Y. D. Kim, U.S. Pat. No. 4,241,044.

13. G. F. Springer and P. R. Desai, Carbohyd. Res., 40 (1975) 183-192.

14. R. Kaifu and T. Osawa, Carbohyd. Res., 69 (1979) 79-88.

15. R. U. Lemieux, D. R. Bundle and D. A. Baker, U.S. Pat. No.4,137,401.

16. R. U. Lemieux, D. A. Baker and D. R. Bundle, Can. J. Biochem., 55(1977) 507-512.

17. H. Paulsen, C. Kolar and W. Stenzel, Angew. Chem. Int. Ed., 15(1976) 440-441.

18. R. U. Lemieux and R. M. Ratcliffe, U.S. Pat. No. 4,195,174.

19. R. U. Lemieux, D. R. Bundle and D. A. Baker, J. Amer. Chem. Soc., 97(1975) 4076-4083.

20. H. H. Sunson, F. Stratton and G. W. Mullard, Brit. J. Haematol., 18(1970) 309-316.

21. H. H. Weetall in Methods in Enzymology, Vol. XLIV, Ed. K. Mosbach,Academic Press, New York, 1976, p. 140.

22. V. E. Jones and S. Leskowitz, Nature, 207 (1965) 596-597.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. The α-glycoside producthaving the structure ##STR5## where n=3-19 and R is an alkoxy, aryloxy,NHNH₂, OH or N₃ group.
 2. The α-glycoside product of claim 1 which is8-methoxycarbonyloctyl-2-acetamido-2-deoxy-α-D-galactopyranoside.
 3. Aprocess to prepare the α-glycoside product of claim 1 which comprises(a)reacting a halide salt of3,4,6-tri-O-acyl-2-azido-2-deoxy-α-D-galactopyranose with amono-hydroxycarboxylate of the general formula HO(CH₂)_(n) COR whereinn=3-19 and R is an alkoxy or aryloxy group; in the presence of apromoter effective to produce the α-glycoside in a suitable solvent; and(b) in any order reducing and N-acetylating the azide group to anacetamido group and removing the O-acyl groups to produce the product ofclaim
 1. 4. The process of claim 3 wherein the halide salt is thebromide salt and the promoter is R'₄ NBr wherein R' is a lower alkylgroup.
 5. The process of claim 4, wherein:the monohydroxycarboxylate ofstep (a) is 8-methoxycarbonyloctanol.
 6. The process of claim 4,wherein:the galacetopyranosyl bromide of step (a) is3,4,6-tri-O-acetyl-2-azido-2-deoxy-α-D-galactopyranosyl bromide.
 7. Theprocess as set forth in claim 4, wherein:the R₄ 'NBr compound of step(a) is tetraethylammonium bromide.
 8. The process of claim 3 wherein thehalide salt is the chloride salt and the promoter is mercuric cyanide.9. The process as set forth in claim 8, wherein:themonohydroxycarboxylate of step (a) is 8-methoxycarbonyloctanol.
 10. Theprocess set forth in claim 8, wherein:the galactopyranosyl chloride ofstep (a) is 3,4,5-tri-O-acetyl-2-azido-2-deoxy-β-D-galactopyranosylchloride.
 11. The process as set forth in claim 3, which furthercomprises:(c) selectively blocking the 4,6-O-hydroxyl groups of theO-α-glycoside; (d) reacting the blocked glycoside with a2,3,4,6-tetra-O-acyl-α-galactopyranosyl halide in the presence of apromoter, in a suitable solvent, to form a β-D-anomeric glycosidiclinkage between the galactopyranosyl halide and the 3-hydroxyl group ofthe blocked glycoside; and (e) removing the O-protecting groups on bothsugars to produce the T-antigenic determinant hapten having thestructure: ##STR6##
 12. The process as set forth in claim 11,wherein:the monohydroxycarboxylate of step (a) is8-methoxycarbonyloctanol.
 13. The process as set forth in claim 11,wherein:the galactopyranosyl halide of step (d) is2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide.
 14. The process asset forth in claim 11, wherein:the blocking group made in step (c) is anacetal.
 15. The process as set forth in claim 11, wherein:themonohydroxylate of step (a) is 8-methoxycarbonyloctanol; thegalactopyranosyl halide of step (d) is2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide; and, the blockinggroup used in step (c) is an acetal.